PROJECT SUMMARY Delivery of nucleic acids (e.g. genes or RNAi) to combat metastatic ovarian cancer is a highly promising therapeutic approach. Tumor cells may be killed by nucleic acids encoding either pro-apoptotic factors or enzymes that can convert prodrugs into toxic molecules. Invariably, however, the success of any gene therapy approach hinges on the ability to deliver the nucleic acid-based effector molecules to target tumors with high specificity and efficiency, a feat that has been largely difficult to achieve. Most vector targeting approaches to date have relied on cell surface receptors overexpressed on some subpopulation of target cancer cells. Unfortunately, there is no unique cell surface biomarker that specifically identifies all cells in a tumor. To overcome this limitation, we propose to develop a platform of protease-activatable viral vectors that we call Provectors. The Provectors cannot deliver transgenes until they become activated by extracellular proteases present at high levels in ovarian tumor microenvironments. In particular, the Provectors are designed to detect matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, whose overexpression is correlated to ovarian cancer progression and death. Our Provector technology is based on the clinically promising adeno-associated virus (AAV), which has recently been approved as the first human gene therapy product in Europe. We have key pilot data demonstrating our ability to build Provectors whose transduction capabilities are activated by MMPs. In an orthotopic ovarian cancer model, a Provector prototype is able to significantly increase transgene delivery and expression in tumors with decreased off-target delivery to liver and spleen. Furthermore, after just a single intravenous injection of Provector encoding HSV-tk, metastatic ovarian tumor bearing mice treated with ganciclovir had significantly better therapeutic outcomes compared to controls. The proposed project will support the design and characterization of the 2nd generation of Provectors with improved features and enable further in vivo testing. In specific aim 1, we will construct a modular platform of high efficiency Provectors for targeting metastatic ovarian tumors. In specific aim 2, we will characterize the developed Provectors via a panel of in vitro assays. Finally, in specific aim 3, we will test the performance of Provectors in preclinical models of ovarian cancer. The proposed project, if successful, will generate a suite of protease-activatable Provectors with improved properties that can target and eradicate metastatic ovarian tumors in vivo.